Nano-Ghosts: Biomimetic membranal vesicles, technology and characterization.

Currently, nano-carriers for anti-cancer drug delivery are complex systems, which struggle with immunogenicity and enhanced permeability effect (EPR)-related problems that halt the clinical translation of many therapeutics. Consequently, a rapidly growing field of research has been focusing on biomimetic nano-vesicles (BNVs) as an effective delivery alternative. Nevertheless, the translation of many BNVs is limited due to scalability problems, inconsistent production process, and insufficient loading efficiency. Here we discuss the process of our previously published BNVs, termed Nano-Ghosts (NGs), which are produced from the membrane of mesenchymal stem cells. We demonstrate the flexibility of the process, while alternating physical methodologies (sonication or extrusion) to produce the NGs while preserving their desired characteristics. We also show that our NGs can be labeled using multiple methods (fluorescence, radiolabeling, and genetic engineering) for tracking and diagnostic purposes. Lastly, we demonstrate that the loading efficiency can be improved by using electroporation to accommodate a range of therapeutics (small molecules, peptides and DNA) that can be delivered by the NGs. Our results emphasize the robustness of the NGs technology, its versatility and a vast range of applications, differentiating it from other BNVs and leading the way towards clinical translation.

Cell-specific cargo delivery using synthetic bacterial spores.

Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. We recently reported the assembly of biocompatible bacterial spore-like particles, termed "SSHELs." Here, we modify SSHELs with an affibody directed against HER2 and load them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduce tumor growth and increase survival with lower toxicity in a mouse tumor xenograft model compared with free drug and with liposomal doxorubicin by preferentially accumulating in the tumor mass. Target cells actively internalize and then traffic bound SSHELs to acidic compartments, whereupon the cargo is released to the cytosol in a pH-dependent manner. We propose that SSHELs represent a versatile strategy for targeted drug delivery, especially in cancer settings.

PLGA Nanoparticles Grafted with Hyaluronic Acid to Improve Site-Specificity and Drug Dose Delivery in Osteoarthritis Nanotherapy.

Nanoparticles (NPs) have a tremendous potential in medicinal applications, and recent studies have pushed the boundaries in nanotherapy, including in osteoarthritis treatments. The aim of this study was to develop new poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) surfaces decorated with hyaluronic acid (HA) to enhance targeted drug specificity to the osteoarthritic knee joint. HA was selected since it binds to specific receptors expressed in many cells, such as the cluster determinant 44 (CD44), a major receptor of chondrocytes, and because of its function in the synovial fluid (SF), such as maintenance of high fluid viscosity. The PLGA polymer was grafted to sodium hyaluronate using dimethoxy-PEG (PLGA-HA) and compared with control PLGA NPs (not grafted). NPs were characterized by 1H-NMR and IR spectroscopy. Then, near-infrared (NIR) dye and gold (20 nm) were encapsulated in the formulated NPs and used to access NPs' performance in in vitro, in vivo, and ex vivo experiments. To test the NPs' CD44 receptor specificity, an antibody assay was performed. All NPs presented a size in the range viable for cell-uptake, no cytotoxicity to chondrocytes was registered. Although all the NPs had a high capacity to be absorbed by the cells, PLGA-HA NPs showed significantly higher affinity towards the chondrocytic C28/I2 cell line. In conclusion, PLGA NPs grafted to sodium hyaluronate showed increased binding to cartilage cells and tissue and enhanced accumulation at the target site. Thus, this study presents a safe drug-delivery system with improved receptor specificity, which may represent an advantageous alternative to current nanotherapies.

Nano-ghosts: Novel biomimetic nano-vesicles for the delivery of antisense oligonucleotides.

Antisense oligonucleotides (ASOs) carry an enormous therapeutic potential in different research areas, however, the lack of appropriate carriers for their delivery to the target tissues is hampering their clinical translation. The present study investigates the application of novel biomimetic nano-vesicles, Nano-Ghosts (NGs), for the delivery of ASOs to human mesenchymal stem cells (MSCs), using a microRNA inhibitor (antimiR) against miR-221 as proof-of-concept. The integration of this approach with a hyaluronic acid-fibrin (HA-FB) hydrogel scaffold is also studied, thus expanding the potential of NGs applications in regenerative medicine. The study shows robust antimiR encapsulation in the NGs using electroporation and the NGs ability to be internalized in MSCs and to deliver their cargo while avoiding endo-lysosomal degradation. This leads to rapid and strong knock-down of miR-221 in hMSCs in vitro, both in 2D and 3D hydrogel culture conditions (>90% and > 80% silencing efficiency, respectively). Finally, in vivo studies performed with an osteochondral defect model demonstrate the NGs ability to effectively deliver antimiR to endogenous cells. Altogether, these results prove that the NGs can operate as stand-alone system or as integrated platform in combination with scaffolds for the delivery of ASOs for a wide range of applications in drug delivery and regenerative medicine.

Drug delivery in intervertebral disc degeneration and osteoarthritis: Selecting the optimal platform for the delivery of disease-modifying agents.

Osteoarthritis (OA) and intervertebral disc degeneration (IVDD) as major cause of chronic low back pain represent the most common degenerative joint pathologies and are leading causes of pain and disability in adults. Articular cartilage (AC) and intervertebral discs are cartilaginous tissues with a similar biochemical composition and pathophysiological aspects of degeneration. Although treatments directed at reversing these conditions are yet to be developed, many promising disease-modifying drug candidates are currently under investigation. Given the localized nature of these chronic diseases, drug delivery systems have the potential to enhance therapeutic outcomes by providing controlled and targeted release of bioactives, minimizing the number of injections needed and increasing drug concentration in the affected areas. This review provides a comprehensive overview of the currently most promising disease-modifying drugs as well as potential drug delivery systems for OA and IVDD therapy.

Pretreating Mesenchymal Stem Cells with Cancer Conditioned-Media or Proinflammatory Cytokines Changes the Tumor and Immune Targeting by Nanoghosts Derived from these Cells.

Nanoghosts (NGs) are nanovesicles reconstructed from the cytoplasmic membranes of mesenchymal stem cells (MSCs). By retaining MSC membranes, the NGs retain the ability of these cells to home in on multiple tumors, laying the foundations, thereby, for the development of a targeted drug delivery platform. The susceptibility of MSCs to functional changes, following their exposure to cytokines or cancer-derived conditioned-media (CM), presents the opportunity to modify the NGs by conditioning their source cells. This opportunity is investigated by comparing the membrane protein composition and the tumor uptake of NGs derived from naïve MSCs (N-NG) against conditioned NGs made from MSCs pre-treated with conditioned-media (CM-NG) or with a mix of the proinflammatory cytokines TNF-α and IL-1ß (Cyto-NG). CM-NGs are found to be more targeted towards immune cells than Cyto- or N-NGs, while Cyto-NGs are the most tumor-targeted ones, with similar immune-targeting capacity as N-NGs but with a higher affinity towards endothelial cells. Proteomic variations were wider in the CM-NGs, with exceptionally higher levels of ICAM-1 compared to N- and Cyto-NGs. From a translational point of view, the data show that the tumor-targeting ability of the NGs, and possibly that of other MSC-derived extracellular vesicles, can be enhanced by simple conditioning of their source cells.